Technical Field
The present disclosure relates to the in vitro differentiation of memory B cells to plasmablasts and plasma cells and genetic modification of these cells to express a protein of interest, such as a specific antibody or other protein therapeutic.
Description of the Related Art
After leaving the bone marrow, the B cell acts as an antigen presenting cell (APC) and internalizes antigens. Antigen is taken up by the B cell through receptor-mediated endocytosis and processed. Antigen is processed into antigenic peptides, loaded onto MHC II molecules, and presented on the B cell extracellular surface to CD4+ T helper cells. These T cells bind to the MHC II/antigen molecule and cause activation of the B cell. Upon stimulation by a T cell, the activated B cell begins to differentiate into more specialized cells. Germinal center B cells may differentiate into memory B cells or plasma cells. Most of these B cells will become plasmablasts, and eventually plasma cells, and begin producing large volumes of antibodies (see e.g., Trends Immunol. 2009 June; 30(6): 277-285; Nature Reviews, 2005, 5:231-242). The most immature blood cell that is considered a plasma cell instead of a B cell is the plasmablast. Plasmablasts secrete more antibodies than B cells, but less than plasma cells. They divide rapidly and are still capable of internalizing antigens and presenting them to T cells. A cell may stay in this state for several days, and then either die or irrevocably differentiate into a mature, fully differentiated plasma cell.
Terminally differentiated plasma cells express relatively few surface antigens, and do not express common pan-B cell markers, such as CD19 and CD20. Instead, plasma cells are identified through flow cytometry by their additional expression of CD38, CD78, the Interleukin-6 receptor and lack of expression of CD45. In humans, CD27 is a good marker for plasma cells, naive B cells are CD27−, memory B-cells are CD27+ and plasma cells are CD27++. CD38 and CD138 are expressed at high levels on plasma cells (See Wikipedia, The Free Encyclopedia., “Plasma cell” Page Version ID: 404969441; Date of last revision: 30 Dec. 2010 09:54 UTC, retrieved Jan. 4, 2011; See also: Jourdan et al. Blood. 2009 Dec. 10; 114(25):5173-81; Trends Immunol. 2009 June; 30(6): 277-285; Nature Reviews, 2005, 5:231-242; Nature Med. 2010, 16:123-129; Neuberger, M. S.; Honjo, T.; Alt, Frederick W. (2004). Molecular biology of B cells. Amsterdam: Elsevier, pp. 189-191; Bertil Glader; Greer, John G.; John Foerster; Rodgers, George G.; Paraskevas, Frixos (2008). Wintrobe's Clinical Hematology, 2-Vol. Set. Hagerstwon, Md.: Lippincott Williams & Wilkins. pp. 347; Walport, Mark; Murphy, Kenneth; Janeway, Charles; Travers, Paul J. (2008). Janeway's immunobiology. New York: Garland Science, pp. 387-388; Rawstron AC (May 2006). “Immunophenotyping of plasma cells”. Curr Protoc Cytom).
Conventional pseudotype retroviruses have demonstrated insufficient infectivity of various tissues and cells. For example, a variety of stem cells including hematopoietic stem cell, and resting B and T cells can be important target cells in gene therapy or the like (Y. Hanazono, Molecular Medicine, Vol. 36, No. 7, 1999), but most of these cell types are found in a nondividing state (Abkowitz, J. L. et al., Nat Med, 2 (2), 190-7, 1996). In general, it is difficult to introduce genes using the retroviral vector exhibiting low infectivity against such nondividing cells.
Resting T and B cells can be efficiently transduced with retroviral vectors pseudotyped with measles virus, glycoproteins, H and F, on their surface (see e.g., Blood 2009 Oct. 8; 114(15):3173-80; Blood 2008 112:4843-4852). However, there are conflicting reports in the literature with regard to the ability of VSV-G pseudotyped viral vectors to transduce B cells (see e.g., Bovia et al., 2003 101:1727-1733; Serafini et al., Virology 325 (2004) 413-424).
Modifying nondividing cells is of particular importance for gene therapy and immunotherapy. However, the ability to differentiate and activate B cells in vitro is an important step in preparing modified B cells for infusion into subjects. Activated and dividing B cells are also not easily genetically modified. Thus, there remains a need in the art to improve the methods and reagents to efficiently genetically modify B cells in vitro. The present disclosure provides vectors and methods for modifying and differentiating/activating B cells such that they can be used effectively in prophylactic and therapeutic applications. The present disclosure provides this and other advantages as described in the detailed description.